CN102168097A - Gene for encoding protein capable of improving heat resistances of plants and microorganisms and application thereof - Google Patents

Abstract

The invention relates to the field of plant gene engineering and in particular relates to a gene for encoding a protein capable of improving heat resistances of plants and microorganisms, the protein encoded by the gene and application of the gene, solving the technical field of providing the genes for encoding the protein capable of improving heat resistances of the plants and the microorganisms. The gene for encoding the protein capable of improving the heat resistances of the plants and the microorganisms contains a nucleotide sequence for encoding the following peptide section: N'-CRICQE X7-45 PCAC X6 AHR X1 CVQ X13-27-C' in which X is any amino acid, and the subscript of X represents the number of amino acid residues. According to the invention, a reserved gene for effectively improving the heat resistances of the plants and the microorganisms is provided for the field and has a good application prospect.

Description

Coding improves plant and the stable on heating proteinic gene of microorganism and uses thereof

Technical field

The present invention relates to plant genetic engineering field, raising plant and stable on heating proteinic gene of microorganism and coded protein and purposes thereof are specifically related to encode.

Background technology

Because the carbonic acid gas free air delivery increases, thereby the Greenhouse effect of the earth constantly aggravate, and cause global climate to warm, and estimate that following 100 years global temperature on average may rise 1.4-5.8 ℃.Global climate warming causes agroecological environment to worsen day by day.Scholarly forecast: climate warming may make crop failure 17%.IRRI (International Rice Research Institute) studies confirm that: 1998-2003, temperature have raise 1 ℃, and output has reduced by 10%.In China, brainstrust thinks that to the year two thousand fifty, national temperature on average will rise 2.2 ℃.Growing plants all is subjected to the influence that temperature raises under physical environment, makes the growth of plant obstacle occur, and particularly some crop sown in spring between heading, filling stage, are easy to be subjected to the influence of hot weather as paddy rice, corn etc., cause the underproduction of farm crop.On the other hand, analyze according to FAO (international food and agricultural organization), to the year two thousand fifty, world population will break through 10,000,000,000.Along with the further increase of population, the population pressure that agricultural faces further increases, and worldwide food shortage situation is with long-term existence.Be subjected to the influence of global warming, retardation of growth even death can appear in a large amount of herbaceous plant, thereby destroy the eubiosis.Therefore, various countries scientific circles are all striving to find the genes involved that improves the vegetable hot tolerance.Up to now, only find that the heat shock protein gene of minority and their transcription factor with heat-resisting relevant, do not see the report that any one independent gene can increase bacterium and plant heat resistance property.This area is badly in need of seeking the thermotolerance gene that can improve plant at present.

Ubiquitin is the polypeptide of a high conservative of being made up of 76 amino-acid residues, gains the name because of it is distributed widely in the various types of cells, will be discerned specifically and be degraded rapidly by the protein of ubiquitin mark.Studies show that the protein degradation that ubiquitin mediation causes is that organism is kept the important physiological and biochemical procedure that self normal growth is grown and grown under adverse environmental factor.

Proteinic ubiquitin modification mainly occurs in the side chain of lysine residue, and multimerization process normally.Can be discerned by proteasome and then be degraded by the protein of many ubiquitinization modification.The enzyme of three kinds of keys has mediated this many ubiquitinization process jointly, comprises ubiquitin activating enzyme E1, ubiquitin binding enzyme E2 and ubiquitin ligase E3.Now basic research is clear for their mechanism of action: at first formed thioester bond and activated single free ubiquitin (this step needs ATP) by the Cys residue and the C end of ubiquitin of E1 by its active centre, E1 is submitted to E2 with the activatory ubiquitin then, at last raise special substrate and E2, and the mediation ubiquitin is transferred to target protein from E2 by E3.Owing to the albumen of proteasome identification ubiquitinization and with its degraded is a nonspecific process, and therefore, E3 has brought into play crucial effects in whole proteinic degradation process, has determined the specificity of reaction.Generally, the ubiquitin reaction comprises an E1, a few E2 and numerous E3.The E3 family that has now found that mainly contains two classes, comprises the RING-finger family and the HECT family that occupy the majority.

Summary of the invention

The technical problem to be solved in the present invention provides a kind of can the coding and improves the gene (abbreviating heat-resisting gene among the present invention sometimes as) of plant and the stable on heating protein of microorganism (abbreviating thermostable protein among the present invention sometimes as).

This coding raising plant or the proteinic gene of microorganism thermotolerance contain the nucleotide sequence of the following peptide section of encoding:

N '-CRICQE X _7-45PCAC X ₆AHR X ₁CVQ X _13-27Structure shown in the-C ' (SEQ ID NO:68), wherein X is an arbitrary amino acid, subscript is represented the quantity of amino-acid residue.

Further, the nucleotides sequence of this peptide section is classified as:

N '-CRICQEED X _3-20NL X _3-20PCAC X ₂SLK X ₁AHR X ₁CVQRWC X _10-24-C ' (SEQ ID NO:69), wherein X is an arbitrary amino acid residue, subscript is represented the quantity of amino-acid residue.

Wherein, above-mentioned protein be ubiquitin ligase (Ubiquitin ligase, E3).

Wherein, above-mentioned protein is transmembrane protein, also contains and strides the film district.

Wherein, said gene derives from the genome of plant.

Wherein, above-mentioned plant is Arabidopis thaliana, paddy rice, corn (Zea mays L.) or castor-oil plant.

Wherein, said gene (1): have the nucleotide sequence shown in SEQ ID NO:39, SEQ ID NO:1, SEQ ID NO:45, SEQ IDNO:47 or the SEQ ID NO:57;

Perhaps (2): said gene has in (1) described arbitrary nucleotide sequence through replacing, lack or add the derive nucleotide sequence of gained of at least one Nucleotide, and the protein of coding with same or analogous function.

Further, described gene has (1): described gene has the nucleotide sequence shown among among among among the SEQ ID NO:57 the 158th～1018, SEQID NO:45 the 96th～848, SEQ ID NO:47 291～1127 or the SEQ ID NO:39 the 68th～946;

Perhaps (2): described gene has in (1) described arbitrary nucleotide sequence through replacing, lack or add the derive nucleotide sequence of gained of at least one Nucleotide, and the protein of coding with same or analogous function.

The present invention provides above-mentioned encoding gene encoded protein matter simultaneously.

The present invention also provides a class to improve plant or the stable on heating protein of microorganism.This proteinoid contains following peptide section:

N '-CRICQE X _7-45PCAC X ₆AHR X ₁CVQ X _13-27Structure shown in the-C ', wherein X is an arbitrary amino acid, subscript is represented the quantity of amino-acid residue.

Further, the nucleotides sequence of this peptide section is classified as:

N '-CRICQEED X _3-20NL X _3-20PCAC X ₂SLK X ₁AHR X ₁CVQRWC X _10-24-C ', wherein X is an arbitrary amino acid.

Wherein, above-mentioned albumen also contains strides the film district, is transmembrane protein.Above-mentioned albumen contains 1-6 and strides the film district.Further, described albumen contains the individual film district of striding of 2-3.

Wherein, the above-mentioned structure of striding the film district is N '-A X _2-6CRS X _2-8LIL X _2-4LL X _1-4LR X _1-10-C ' (SEQ IDNO:70), perhaps N '-L X _2-4R X _1-5GFLL X _1-7YIMAW X _1-15At least a shown in the-C ' (SEQ ID NO:71), wherein X is an arbitrary amino acid, subscript is represented the quantity of amino-acid residue.Wherein, above-mentioned each striden and had one section between the film district and flexibly connect.Wherein, hold near C in the above-mentioned film district of striding.Wherein, the above-mentioned film district of striding directly is connected by the section of flexibly connecting with zinc finger domain.Wherein, above-mentioned albumen also has signal peptide at the N end.Wherein, above-mentioned signal peptide is the film localization signal peptide.

Wherein, above-mentioned protein is ubiquitin ligase Ubiquitin ligase.

Wherein, above-mentioned protein source is in plant.

Wherein, above-mentioned albumen has by SEQ ID NO:1 (its amino acid sequence coded is seen SEQ ID NO:2), the 158th～1018 (its amino acid sequence coded is seen SEQ ID NO:58) among the SEQ ID NO:57, the 96th～848 (its amino acid sequence coded is seen SEQ ID NO:46) among the SEQ ID NO:45,291～1127 (its amino acid sequence coded is seen SEQ ID NO:48) among the SEQ ID NO:47 or the coded aminoacid sequence that obtains of nucleotide sequence shown in the 68th～946 (its amino acid sequence coded is seen SEQ ID NO:40) among the SEQ ID NO:39;

Perhaps (2): described gene has process replacement in (1) described arbitrary aminoacid sequence, lacks or add the aminoacid sequence of at least one amino acid derived gained, and has same or analogous function.

The present invention also provides the gene of the above-mentioned thermostable protein of encoding.

The present invention provides one section peptide section in addition.Described peptide section contains N '-CRICQE X _7-45PCAC X ₆AHR X ₁CVQ X _13-27Structural domain shown in the-C ', wherein X is an arbitrary amino acid, index number is represented the quantity of amino-acid residue.

Further, the aminoacid sequence of this structural domain is:

N '-CRICQEED X _3-20NL X _3-20PCAC X ₂SLK X ₁AHR X ₁CVQRWC X _10-24Structural domain shown in the-C ', wherein X is an arbitrary amino acid residue, index number is the quantity of amino-acid residue.

Wherein, above-mentioned peptide section derives from plant.Described plant can be Arabidopis thaliana, paddy rice, corn or castor-oil plant.

Wherein, above-mentioned peptide section can form zinc fingers.Be N '-CRICQE X _7-45PCAC X6 AHR X1 CVQ X _13-27-C ' or N '-CRICQEED X _3-20NL X _3-20PCAC X ₂SLK X ₁AHR X ₁CVQRWC X _10-24Structural domain shown in the-C ' is for forming the structural domain of zinc fingers, and wherein X is an arbitrary amino acid, and index number is represented the quantity of amino-acid residue.

Wherein, above-mentioned peptide section can be given protein raising plant or the stable on heating function of microorganism.

Further, above-described resistance is thermotolerance.Further, described protein is ubiquitin ligase.

Further, above-mentioned peptide section has following peptide sequence:

(1): the aminoacid sequence shown in SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66 or SEQ ID NO:67;

Perhaps, process replaces, lacks or add the aminoacid sequence of at least one amino acid derived gained in (1) described arbitrary amino acid sequence of polypeptide, and has same or analogous function.

Wherein, above-mentioned " at least one " is meant one or several (in 10).Above-mentioned " same or analogous function " is meant the thermotolerance that can improve plant or microorganism.

The present invention provides the nucleotide sequence of the above-mentioned peptide section of encoding in addition.

It is the method for the gene of coding thermostable protein that the present invention provides the nonrefractory proteinic genetic modification of will encoding in addition, and its step is added into the nonrefractory proteinic gene of coding or replacement one section nucleotide sequence wherein for the coding nucleotide sequence with above-mentioned peptide section.Certainly such interpolation or replacement be occur between the codon but not within so that whole improved gene still has the ability of coding whole protein.Promptly, the nucleotide sequence that steps of the method are the peptide section that coding is above-mentioned is added in the nonrefractory proteinic gene of encoding or replaces the nonrefractory proteinic gene of coding one section nucleotide sequence wherein, described interpolation or replacement are to occur between the codon, so that whole improved gene still has the ability of coding whole protein.

Wherein, the above-mentioned one section nucleotides sequence that is replaced is classified the nucleotide sequence of coding Zinc finger domain as.It is the nucleotide sequence that one section nucleotides sequence being replaced in the nonrefractory proteinic gene of above-mentioned coding is classified the coding Zinc finger domain as.

Wherein, above-mentioned nonrefractory protein is ubiquitin ligase Ubiquitin ligase (E3 ligase enzyme).

On the basis of foregoing invention, the present invention also provide a kind of improve plant heat resistance property or preparation high heat resistance plant method.This method may further comprise the steps:

A, the gene of above-mentioned coding thermostable protein operationally is connected in expression regulation sequence on the carrier after, formation can be expressed the recombinant vectors of this gene;

B, change the recombinant vectors among the step a over to vegetable cell;

C, obtain transformant through screening, then transformant is cultivated into transfer-gen plant or its offspring, described offspring comprises plant seed or plant tissue.Further, the resistance in the aforesaid method is thermotolerance.

The present invention also provides the above-mentioned gene order or the carrier of nucleotide sequence.Wherein, described carrier is an expression vector.

Further, described expression vector is a carrier for expression of eukaryon.

The present invention also provides the host cell that contains above-mentioned described carrier.Described host cell is mainly vegetable cell or microorganism.

Recombinant vectors of the present invention is gene to be inserted in the carrier obtain, and above-mentioned carrier can be selected various carrier, especially carrier for expression of eukaryon known in the art (as pBI121 or pCAMBIA2301) for use.The present invention can transform host plant cell with above-mentioned recombinant vectors, and screening obtains transformant.Use various above-mentioned adverse environmental factors to screen then.

In the present invention, the gene order that is similar to " the nucleotide sequence process in SEQ ID NO:1 replaces, lacks or add at least one Nucleotide derived sequence " statement is meant that generally coding has the nucleotide sequence and the degenerate sequence thereof of the polypeptide of the coded protein-active of SEQ ID NO:1.This degenerate sequence be meant have one or more codons to be encoded in the described sequence degenerate codon of same amino acid replaces the back and the sequence that produces.Because the degeneracy of codon, so be low to moderate about 89% the degenerate sequence described sequence of SEQ ID NO:1 of also encoding out with SEQ ID NO:1 homology.In addition, the implication of " nucleotide sequence in SEQ ID NO:1 through replace, lack or add at least one Nucleotide derived sequence " also comprises can be under the rigorous condition of moderate, better under highly rigorous condition with the nucleotide sequence of SEQ ID NO:1 nucleotide sequence hybridization.This term also comprise with SEQ ID NO:1 in homology of nucleotide sequence at least 80%, preferably at least 89%, more preferably at least 90%, at least 95% nucleotide sequence best.Identical function in the present invention is meant the thermotolerance that improves plant or microorganism.

This term also comprises encoding to have the variant form of open reading frame sequence among the proteic SEQ ID NO:1 with natural SEQ ID NO:1 identical function.These variant forms comprise (but being not limited to): several (are generally 1～90, preferably 1～60, more preferably 1～20,1～10 best) disappearance, insertion and/or the replacement of Nucleotide, and several (are generally in 60 to hold interpolation 5 ' and/or 3 ', preferably being in 30, more preferably is in 10, is in 5 best) Nucleotide.

In the present invention, the statement of " process replaces, lacks or add the aminoacid sequence of at least one amino acid derived gained in described aminoacid sequence " includes, but are not limited to several and (is generally 1-50, preferably 1-30, more preferably 1-20,1-10 best) amino acid whose disappearance, insertion and/or replacement, and at C-terminal and/or N-terminal interpolation one or several (being generally in 20, preferably is in 10, more preferably is in 5) amino acid.For example, in described albumen, when replacing, can not change proteinic function usually with the close or similar amino acid of performance.Again such as, add one or several amino acid at C-terminal and/or N-terminal and also can not change proteinic function usually.This term also comprises described proteic active fragments and reactive derivative.Identical function in the present invention is meant the thermotolerance that improves plant or gives other protein and improve plant or the stable on heating function of microorganism.

The statement of " process replaces, lacks or add the aminoacid sequence of at least one amino acid derived gained in described aminoacid sequence " has also included, but are not limited to 10 at the most, preferably at the most 8, more preferably 5 amino acid is replaced by similar performance or close amino acid and is formed polypeptide, i.e. conservative property variation polypeptide at the most.These conservative property variation polypeptide are preferably replaced according to table 1 and are produced.

Table 1 amino acid substitution table

Initial residue	Representational replacement	The preferred replacement
			Ala(A)	Val；Leu；Ile	Val
Arg(R)	Lys；Gln；Asn	Lys
			Asn(N)	Gln；His；Lys；Arg	Gln
Asp(D)	Glu	Glu
			Cys(C)	Ser	Ser
Gln(Q)	Asn	Asn
			Glu(E)	Asp	Asp
Gly(G)	?Pro；Ala	Ala
			His(H)	?Asn；Gln；Lys；Arg	Arg
Ile(I)	?Leu；Val；Met；Ala；Phe	Leu
			Leu(L)	?Ile；Val；Met；Ala；Phe	Ile
Lys(K)	?Arg；Gln；Asn	Arg
			Met(M)	?Leu；Phe；Ile	Leu
Phe(F)	?Leu；Val；Ile；Ala；Tyr	Leu
			Pro(P)	?Ala	Ala
Ser(S)	?Thr	Thr
			Thr(T)	?Ser	Ser
Trp(W)	?Tyr；Phe	Tyr
			Tyr(Y)	?Trp；Phe；Thr；Ser	Phe
Val(V)	?Ile；Leu；Met；Phe；Ala	Leu

The present invention also comprises the analogue of albumen required for protection or polypeptide.The difference of these analogues and natural SEQ ID NO:2 polypeptide can be the difference on the aminoacid sequence, also can be the difference that does not influence on the modified forms of sequence, perhaps haves both at the same time.These polypeptide comprise natural or the inductive genetic variant.The induce variation body can obtain by various technology, as by radiation or be exposed to mutagenic compound and produce random mutagenesis, also can pass through site-directed mutagenesis method or the biological technology of other known moleculars.Analogue also comprises having the analogue that is different from the amino acid whose residue of natural L-(as D-amino acid), and has non-natural analogue that exist or synthetic amino acid (as β, gamma-amino acid).Should be understood that albumen of the present invention or peptide section are not limited to above-mentioned representational albumen or the polypeptide that exemplifies.

(the not changing primary structure usually) form of modification comprises: the chemically derived form such as the acetylize or carboxylated of the polypeptide that body is interior or external.Modification also comprises glycosylation, carries out glycosylation modified and polypeptide that produce in the procedure of processing as those in the synthetic and processing of polypeptide or further.This modification can be carried out glycosylated enzyme (as mammiferous glycosylase or deglycosylating enzyme) and finishes by polypeptide is exposed to.Modified forms also comprises have the phosphorylated amino acid residue sequence of (as Tyrosine O-phosphate, phosphoserine, phosphothreonine).Thereby also comprise the polypeptide that has been improved its anti-proteolysis performance or optimized solubility property by modifying.

" operationally being connected in " described in the present invention is expressed as follows situation: promptly some part of linear DNA sequence can influence the activity of same other parts of linear DNA sequence.For example, if signal peptide DNA as precursor expression and participate in the secretion of polypeptide, signal peptide (secretion leader sequence) DNA operationally is connected in polypeptid DNA so; If transcribing of promotor control sequence, it is operationally to be connected in encoding sequence so; When if ribosome bind site is placed in the position that can make its translation, it is operationally to be connected in encoding sequence so.Generally, " operationally being connected in " means adjacent, then means in reading frame adjacent for the secretion leader sequence.

Beneficial effect of the present invention is: the gene of class coding thermostable protein and coded protein thereof mainly are provided, and one class can play the polypeptide and the encoding gene thereof of similar effect, and these genes and the purposes of protein in the thermotolerance that improves plant and microorganism.The present invention has good application prospects for this area provides the stable on heating alternative gene that newly can effectively improve plant and microorganism.

Description of drawings

Fig. 1 changes the colibacillary growth curve of AtTR1 gene and mutator gene thereof.Indicating AtTR1 among the figure is the intestinal bacteria of overexpression AtTR1 gene, and MT1～6 are respectively the intestinal bacteria of overexpression AtTR1 mutator gene, and WT is not genetically modified intestinal bacteria.

Fig. 2 changes the colibacillary growth curve of AtTR1 gene castor-oil plant homologous gene RcTR1.

Fig. 3 changes the colibacillary growth curve of AtTR1 trans-genetic hybrid rice homologous gene OsTR1-1, OsTR1-2.

Fig. 4 changes the colibacillary growth curve of AtTR1 gene corn homologous gene ZmTR1.

Fig. 5 replaces or lacks the colibacillary growth curve that AtTR1 albumen is striden the film district.

Fig. 6 replaces or lacks the colibacillary growth curve that OsTR1-1 albumen is striden the film district.

Fig. 7 replaces or lacks the colibacillary growth curve that ZmTR1 albumen is striden the film district.

The proteic E3 ubiquitin ligase of Fig. 8 AtTR1 is active to detect (+expression adding;-expression does not add).

The proteic E3 ubiquitin ligase of Fig. 9 OsTR1-1 is active to detect (+expression adding;-expression does not add).

The proteic E3 ubiquitin ligase of Figure 10 ZmTR1 is active to detect (+expression adding;-expression does not add).

Embodiment

Among the following embodiment, all unreceipted concrete experiment conditions, be according to normal condition well known to those skilled in the art Sambrook J for example, Russell D.W., 2001, Molecular Cloning:A laboratory manual (3 ^RdEd), the condition described in the Spring Harbor Laboratory Press, or the condition of advising according to manufacturer.Among the following embodiment, used carrier pET28, pGEX-2T, pGEM-T be available from Qiagen company, and bacterial strain BL21 is available from Qiagen company, and bacterial strain EHA105, carrier pBI121 are available from Clontech company.

Using genetic engineering technique that plant is improved is in recent years focus.Use genetic engineering technique in the hope of improving the resistance of plant, cultivating resistance of reverse system also is a feasible road.But, the also rarely seen at present report that the individual gene of the multiple resistance that can comprehensively improve plant and microorganism is arranged.The gene A tTR1 that can improve vegetable hot stress resistance (thermotolerance) that the applicant is cloned into from Arabidopis thaliana.

Discover that described gene A tTR1 expressed proteins has ubiquitin E3 ligase enzyme activity in the ubiquitin protein system; By with AtTR1 gene transformation Arabidopis thaliana, rape, intestinal bacteria, experimental result finds that the thermotolerance of genetically modified organism all increases.This gene can be used for improving plant, microorganism to the pyritous tolerance degree, reduces the underproduction loss that high temperature causes, reduces cost simultaneously, has important economic implications and application prospect.

In order to seek the mechanism of action of AtTR1 gene, search the proteic keying action of AtTR1 site, the more proteic conserved amino acids of AtTR1 are suddenlyd change, study its changes of function situation.

Table 2 AtTR1 and MARCH family protein sequence alignment

Albumen	Close key sequence (ring structure)
		AtTR1	69 CRICQE.EDSTKNLEAPCACNGSLKYAHRKCVQRWCNEKGDITCEIC 114
MARCH2	64 CRICHE.GANGECLLSPCGCTGTLGAVHKSCLEKWLSSSNTSYCELC 109
		MARCH4	163 CRICFQ.GPEQGELLSPCRCDGSVKCTHQPCLIKWISERGCWSCELC 208
MARCH6	9 CRVCRSEGTPEKPLYHPCVCTGSIKFIHQECLVQWLKHSRKEYCELC 55

By protein structure comparison (seeing Table 2), find that MARCH (MembraneAssociated Ring-CH) protein structure of AtTR1 albumen and participant's para-immunity adjusting is similar, all have typical finger ring (Ring-CH) structural domain.Several critical sites shown in the comparison result are carried out rite-directed mutagenesis, and the result shows that the thermotolerance of these mutant changes, and than non-transgenic type thermotolerance or high or low, changes the AtTR1 genotype but all be lower than.Illustrate that these critical sites have determined AtTR1 albumen to improve the functionally active of biological resistance of reverse.

Above-mentioned result of study the basis on, in other plant, found a plurality of homologous genes of AtTR1 gene.These homologous genes have the zone of some high conservatives, also have the effect (the core sequence analysis is referring to table 3) that strengthens plant heat resistance property.

The functional structure site comparison of table 3 AtTR1 and homologous protein thereof

Determine to have resistance toheat at last, should have a conservative RINGv zone so, its aminoacid sequence is as follows:

Further should the zone be:

Italicized item amino acid is the selected site that can not suddenly change of mutant test, can lose after the sudden change or obviously reduces heat-resisting function, and wherein X is arbitrary amino acid (being preferably natural amino acid), and index number is represented the quantity of amino-acid residue.

The pattern list of the common zinc finger protein of table 4

Can find out from table 4,1,2 and 5,6 wherein above-mentioned site amino acid form zinc fingers, and x wherein can be arbitrary amino acid, the peculiar zinc fingers of the 3rd behavior albumen of the present invention (RINGv), peculiar in plant, closely related with the proteic heat resistanceheat resistant function of the present invention.

The structure of the clone of heat-resisting gene A tTR1 and overexpression recombinant vectors in embodiment one Arabidopis thaliana

1, screening obtains a fragment gene AtTR1 from Arabidopis thaliana, and its nucleotide sequence is shown in SEQ ID NO:1 in the sequence table.According to the design of nucleotide sequence shown in SEQ ID NO:1 primer,

Upstream primer (SEQ ID NO:3): 5 '-ATGGCTGATCATTTGAGTTTATGT-3 ',

Downstream primer (SEQ ID NO:4): 5 '-TCAAACTGGTGTTGGGACATTGGATA-3 '.

Then through the PCR nucleotide sequence shown in the SEQ ID NO:1 that from Arabidopis thaliana cDNA, increases.To PCR product purification (seeing Qiagen company disclosed PCR product purification data),, obtain the gene fragment of sequence SEQ ID NO:1 through sequence verification.

2, according to the design of nucleotide sequence shown in SEQ ID NO:1 primer

Upstream primer (SEQ ID NO:5): 5 '-CGCGGATCC ATGGCTGATCATTTGAGTTTATGT-3 ',

Downstream primer (SEQ ID NO:6): 5 '-CCGGAGCTC TCAAACTGGTGTTGGGACATTGGATA-3 '.

Through PCR, the nucleotide sequence shown in the SEQ ID NO:1 that will increase complete from Arabidopis thaliana cDNA amplifies restriction enzyme site.To PCR product purification (seeing the disclosed data of Qiagen company), to cut with BamH1 and Sac1 enzyme then, glue reclaims, and is connected (connection site: BamH1 and Sac1) with carrier pBI121, obtains the overexpression recombinant plasmid that contains SEQ ID NO:1.

The structure of the rite-directed mutagenesis of embodiment two AtTR1 genes and mutator gene overexpression recombinant vectors

1, several sites of picked at random, design comprises the primer (the underscore base is the mutational site) in mutational site according to nucleotide sequence shown in the SEQ ID NO:1, makes up rite-directed mutagenesis sequence (Stratagene company

Site-DirectedMutagenesis Kit).

Protein sequence the 69th amino acids mutant primer:

Upstream primer (SEQ ID NO:7): 5 '-TTCTC CAATC TGTTG AG AGTCGTAT TTGCC-3 '

Downstream primer (SEQ ID NO:8): 5 '-GGCAA ATACG ACTCT CAACA GATTG GAGAA-3 '

Protein sequence the 72nd amino acids mutant primer:

Upstream primer (SEQ ID NO:9): 5 '-AGTGT CGTAT T GGCC AAGAG GAAGA TAGTA CT-3 '

Downstream primer (SEQ ID NO:10): 5 '-AG TACTA TCTTC CTCTT G GCCA ATACG ACACT-3 '

Protein sequence the 85th amino acids mutant primer:

Upstream primer (SEQ ID NO:11): 5 '-CTTGA AGCTC CT AGTGCTTG TAA-3 '

Downstream primer (SEQ ID NO:12): 5 '-TTA CAAGC ACTAG GAGCT TCAAG-3 '

Protein sequence the 87th amino acids mutant primer:

Upstream primer (SEQ ID NO:13): 5 '-GAAGC TCCTT GTGCT CGTAA TGGTA GTTTA AA-3 '

Downstream primer (SEQ ID NO:14): 5 '-TT TAAAC TACCA TT ACGAGCAC AAGGA GCTTC-3 '

Protein sequence the 95th amino acids mutant primer:

Upstream primer (SEQ ID NO:15): 5 '-TAAAG TATGC T AACC GCAAG TGTGT TC-3 '

Downstream primer (SEQ ID NO:16): 5 '-GA ACACA CTTGC G GTTA GCATA CTTTA-3 '

Protein sequence the 98th amino acids mutant primer:

Upstream primer (SEQ ID NO:17): 5 '-ACCGC AAG TA TGTTC AGCGT TGGTG TA-3 '

Downstream primer (SEQ ID NO:18): 5 '-TA CACCA ACGCT GAAC A TACTT GCGGT-3 '

Do template with the carrier pET28 that is connected with SEQ ID NO:1, pcr amplification goes out to contain SEQ ID NO:19 sequence (SEQ IDNO:2 sequence N holds the 69th halfcystine to replace to Serine), SEQ ID NO:20 sequence (SEQ ID NO:2 sequence N holds the 72nd halfcystine to replace to glycine), SEQ ID NO:21 sequence (SEQ ID NO:2 sequence N holds the 85th halfcystine to replace to Serine), SEQ ID NO:22 sequence (SEQ ID NO:2 sequence N holds the 87th halfcystine to replace to arginine), SEQ ID NO:23 sequence (SEQ ID NO:2 sequence N holds the 95th hyte propylhomoserin to replace to l-asparagine), the clone of the mutant plasmid of SEQ ID NO:24 sequence (SEQ ID NO:2 sequence N holds the 98th halfcystine to replace to tyrosine).

2, by amplimer, the nucleotide sequence shown in complete SEQ ID NO:19～24 increases from mutant plasmid.

Upstream primer (SEQ ID NO:5): 5 '-CGCGGATCC ATGGCTGATCATTTGAGTTTATGT-3 ',

Downstream primer (SEQ ID NO:6): 5 '-CCGGAGCTC TCAAACTGGTGTTGGGACATTGGATA-3 '.

To PCR product purification (seeing the disclosed data of Qiagen company), cut with BamH1 and Sac1 enzyme then, glue reclaims, and is connected with carrier pBI 121 (connection site: BamH1 and Sac1), obtain the overexpression recombinant plasmid that contains SEQ ID NO:19～24, be designated as MT1～6.

The colibacillary resistance toheat of embodiment three overexpression AtTR1 and mutator gene thereof is identified

1, transformed into escherichia coli

With the recombinant plasmid transformed intestinal bacteria, coat on the LB solid medium that contains Amp, through sequence verification, obtain containing the intestinal bacteria pET28 bacterial strain of recombinant plasmid.

2, preparation substratum

Preparation LB liquid nutrient medium adds microbiotic Kan 50ug/ml, behind the Cam 50ug/ml, and packing 24 pipes, every pipe 10ml, stand-by.

3, preparation bacterial suspension

Get overexpression AtTR1 and 6 kinds of mutator gene intestinal bacteria (MT1～6) and 37 ℃ of activation of spending the night of the single bacterium colony of non-transgenic intestinal bacteria (totally 8 kinds).

4, drip for the examination bacterium

Insert activation bacterium liquid 0.05ml in every pipe LB liquid nutrient medium, every kind of bacterium connects 3 test tubes, shake up back shaking culture 3 hours (37 ℃, 225rpm).

5, cultivate and observe

Cultivate after 3 hours for 37 ℃, add 0.1mM IPTG (sec.-propyl-β-D-sulfo-galactopyranoside) again, cultivate 10 hours observationss for 42 ℃.Judge overexpression AtTR1 and 6 kinds of mutator gene intestinal bacteria (MT1～6) and the colibacillary growing state of non-transgenic with range estimation, regularly repeatedly measure OD600 value (general the analysing in Beijing led to the TU-1800 of instrument company type ultraviolet spectrophotometer) simultaneously, in order to draw colibacillary growth curve not of the same race, (indicating AtTR1 among the figure is the intestinal bacteria of overexpression AtTR1 gene to the results are shown in Figure 1, MT1～6 are respectively the intestinal bacteria of overexpression AtTR1 mutator gene, and WT is not genetically modified intestinal bacteria).

Found that, the intestinal bacteria of overexpression AtTR1 mutator gene, though show certain thermotolerance, but can find out that its growth curve slope is significantly less than the intestinal bacteria of overexpression AtTR1, after illustrating to undergo mutation in these 6 sites, can cause the heat-resisting function of this gene to decrease, these 6 sites are exercised heat-resisting function in vivo for AtTR1 albumen to be had and vital role.

The resistance toheat of the rape of embodiment four overexpression AtTR1 and mutator gene thereof is identified

1, the method for plumular axis dip-dye transforms swede type rape

Obtaining of 1-1, aseptic seedling

Choose the swede type rape seed of full seed, 4 ℃ of vernalization of spending the night (the maintenance seed germination is synchronous) are taken out then, with 70% alcohol immersion 30s, 0.1% mercuric chloride (HgCl2) solution soaking 8～10 minutes, aseptic water washing 5 times, filter paper blots, and is inoculated on the MS solid medium.Put in the culturing room 24 ℃, secretly cultivated 2-3 days, take out illumination 16h/d then and continue to sprout.Get 5-7 centimetre of (about 7-8 days) aseptic seedling hypocotyl as transformation receptor.

1-2, hypocotylar pre-cultivation

Rape hypocotyls is cut into 7mm left and right sides segment, and being uniformly dispersed places the pre-cultivation of carrying out in the pre-culture medium (MS+2mg/L 6-BA, 1mg/L 2,4-D, 2.5mg/L AgNO3,19.62mg/L Syringylethanone) 2～3 days (visible hypocotyl chap).

1-3, hypocotylar dip-dye and cultivation altogether

Picking contains the Agrobacterium of the overexpression recombinant plasmid of SEQ ID NO:1 and SEQ ID NO:19～24, be inoculated in and contain the 20mg/L Streptomycin sulphate, the 50mg/L kantlex, in the LB liquid nutrient medium of 40mg/L Rifampin, 28 ℃ are shaken the bacterium back of spending the night and collect thalline, be resuspended in the MS liquid nutrient medium that contains the 100mg/L Syringylethanone to OD600=0.4～0.6,28 and ℃ shook bacterium 1-2 hour.

To immerse respectively through the rape hypocotyls of pre-incubated stalwartness in the Agrobacterium bacterium liquid of the overexpression recombinant plasmid that contains SEQ ID NO:1 and SEQ ID NO:19～24 30 seconds～1 minute, during this constantly vibration bacterium liquid is fully contacted with rape hypocotyls.Blot unnecessary bacterium liquid rapidly with aseptic filter paper, rape hypocotyls is lain against on the common substratum (MS+2mg/L 6-BA, 1mg/L2,4-D, 2.5mg/L AgNO3,19.62mg/L Syringylethanone), cultivated altogether 2 days.

Inducing of 1-4, screening and culturing and bud

Seven kinds of rape hypocotyls after cultivating are altogether inserted continuation cultivation in the division culture medium (MS+2mg/L 6-BA, 1mg/L 2,4-D, 2.5mg/L AgNO3,19.62mg/L Syringylethanone) respectively.Per 2 weeks are upgraded substratum once, cultivate for 4 weeks, obtain the callus bud.

1-5, take root

At screening culture medium (MS+2mg/L 6-BA, 2.5mg/L AgNO3, the 500mg/L Pyocianil, the 10mg/L kantlex) treats on that the callus bud grows to when 4～6 true leaves are arranged, bud is downcut from callus, move in the root media (1/2MS, 0.15mg/L NAA, 250mg/L cephamycin).When treating that the regrowth root growth is flourishing, culture tank is moved to outdoor 2～3 days, will cultivate cover then and open, hardening is 2～3 days in culturing room.

1-6, potted plant cultivation

The overexpression transfer-gen plant that will contain SEQ ID NO:1 and SEQ ID NO:19～24 gene orders grows complete root system respectively on root media, it is changed over to potted plant.

2, the PCR of transgene rape detects

After treating that regeneration plant is grown up in the soil, respectively get the total DNA of a small amount of extracting of blade, do template, carry out PCR respectively and detect with the DNA that extracts.

Detect the AtTR1 primer:

Upstream primer (SEQ ID NO:25): 5 '-TGATCATTTGAGTTTATGTACCGA-3 '

Downstream primer (SEQ ID NO:26): 5 '-TCAAACTGGTGTTGGGACATTGGAT-3 '

Detect the MT1 primer:

Upstream primer (SEQ ID NO:27): 5 '-AATCTGTTGAG AGTCGTATTTGCCAA-3 '

Downstream primer (SEQ ID NO:28): 5 '-TCAAACTGGTGTTGGGACATTGGAT-3 '

Detect the MT2 primer:

Upstream primer (SEQ ID NO:29): 5 '-GTCGTATT GGCCAAGAGGAAGATAG-3 '

Downstream primer (SEQ ID NO:30): 5 '-TCAAACTGGTGTTGGGACATTGGAT-3 '

Detect the MT3 primer:

Upstream primer (SEQ ID NO:31): 5 '-CTTGAAGCTCCT AGTGCTTGTAAT-3 '

Downstream primer (SEQ ID NO:32): 5 '-TCAAACTGGTGTTGGGACATTGGAT-3 '

Detect the MT4 primer:

Upstream primer (SEQ ID NO:33): 5 '-CTTGTGCT CGTAATGGTAGTTTAAAGT-3 '

Downstream primer (SEQ ID NO:34): 5 '-TCAAACTGGTGTTGGGACATTGGAT-3 '

Detect the MT5 primer:

Upstream primer (SEQ ID NO:35): 5 '-AAGTATGCT AACCGCAAGTGTGTTCT-3 '

Downstream primer (SEQ ID NO:36): 5 '-TCAAACTGGTGTTGGGACATTGGAT-3 '

Detect the MT6 primer:

Upstream primer (SEQ ID NO:37): 5 '-TATGCTCACCGCAAG TATGTTCAGCGTTT-3 '

Downstream primer (SEQ ID NO:38): 5 '-TCAGACTGGTGTTGGGTTGGATAT-3 '

Whether agarose electrophoresis detects then has target stripe (stripe size is about 860bp) to occur, then represents goal gene to change swede type rape over to if having, and through detecting, has obtained changing over to the rape of corresponding gene.

3, the transgene rape resistance toheat is identified---the seed germination situation under the hot shock condition

3-1, seed vernalization

Choose totally 8 kinds in 7 kinds of transgenic brassica napus seeds (AtTR1, MT1, MT2, MT3, MT4, MT5, MT6) and not genetically modified wild swede type rape seed (WT), every kind each 200, add 4 ℃ of soaked overnight of water.

3-2, heat shock are handled

8 kinds of Semen Brassicae campestriss are respectively charged in the test tube of mark, every kind 2 pipe, 100 of every pipes are divided into two groups of A, B with it, 45 ℃ of water-baths of A group 3 hours, B group soak at room temperature 2 hours.

3-3, result observe

After 2 hours, seed is placed the plate that is covered with filter paper, put into (illumination 6000～the 8000lux of culturing room, the 16h/8h light dark period) cultivates, observed once in per 24 hours, the statistics relative germination rate of seed (percentage of germination after relative germination rate=heat shock/normal temperature percentage of germination * 100%) the results are shown in Table 5.

Relative germination rate (unit: %) after the heat shock of table 5 Semen Brassicae campestris

	AtTR1	MT1	MT2	MT3	MT4	MT5	MT6	WT
									After 1 day	0	0	0	0	0	0	0	0
After 2 days	32.48	20.00	14.68	13.75	10.06	10.11	16.94	12.82
									After 3 days	68.52	41.97	38.55	35.69	30.17	26.96	40.00	32.22
After 4 days	74.13	57.43	41.26	44.20	42.88	40.25	47.86	40.85
									After 5 days	84.00	62.35	47.87	50.00	51.25	47.01	49.96	46.87
After 6 days	86.17	69.02	56.95	62.75	60.77	59.20	62.45	59.50
									After 7 days	93.55	70.38	65.82	70.13	69.00	62.85	68.29	64.00

4, the transgene rape resistance toheat is identified---the seedling growth situation under the hot shock condition

7 kinds of transgenic brassica napus seeds (AtTR1, MT1, MT2, MT3, MT4, MT5, MT6) and not genetically modified wild swede type rape seed (WT) be positioned over respectively on the moistening filter paper sprout, treat to move in the vegetable mould after the broken shell, cultivate about 15 days (two true leaves grow up to) for 22 ℃, change 34 ℃ of heat stresses then over to, condition is sunshine 14h, dark 10h.Observe the growing state of various rapes.

Found that, behind the heat stress 3 days, the swede type rape growth is suppressed, jaundice, wilting phenomenon all appear in swede type rape transgenic line MT1-6 and wild-type WT, the wherein yellow of MT1, MT2, MT6, wilting degree are lighter relatively, WT, MT3, MT4, MT5 are more serious relatively, and the AtTR1 growth is normal.Behind the heat stress 5 days, swede type rape transgenic line MT1-6 and wild-type WT are dead, and AtTR1 remains work, and growth is normal.

The castor-oil plant homologous gene RcTR1 of embodiment five AtTR1 obtains and changes the experiment of the colibacillary thermotolerance of RcTR1

1, the homogenic clone of castor-oil plant and obtaining

Extract the total RNA of castor-oil plant (Invitrogen company's T RIzol reagent) with the TRIzol method, reverse transcription then (promega company reverse transcription test kit) becomes cDNA, respectively according to castor-oil plant RcTR1 nucleotide sequence shown in the SEQ ID NO:39 (its amino acid sequence coded is seen SEQ ID NO:40) design primer:

Upstream primer (SEQ ID NO:41): 5 '-GTGGTGTAGCAGGATTTTTAATC-3 '

Downstream primer (SEQ ID NO:42): 5 '-GCATCCCCATTCATTTCAT-3 '

Through the PCR 37-964 bit sequence in the nucleotide sequence that from castor-oil plant cDNA, increases shown in the SEQ ID NO:39.Purified pcr product (seeing the disclosed data of Qiagen company) through sequence verification, obtains required sequence.

2, be with the primer of restriction enzyme site according to above-mentioned sequence fragment design of increasing, construction recombination plasmid.

Upstream primer (SEQ ID NO:43): 5 '-CCGGAATTCATGAGTGATCAACTAGTTTTG-3 '

Downstream primer (SEQ ID NO:44): 5 '-CCCAAGCTTTCATTGAAGTGGCTCTTG-3 '

Through PCR, from the PCR product of step 1, amplify shown in the SEQ ID NO:39 68-946 bit sequence (its amino acid sequence coded is seen SEQ ID NO:40) in the nucleotide sequence.Purified pcr product (seeing the disclosed data of Qiagen company) is cut with EcoR1 and HindIII enzyme then, and glue is connected with carrier pET28 respectively after reclaiming fragment, obtains recombinant plasmid.Recombinant plasmid is changed in the e. coli jm109, coat on the LB solid medium that contains kantlex,, obtain containing the e. coli jm109 bacterial strain of recombinant plasmid through sequence verification.

3, after recombinant plasmid is bred in the e. coli jm109 bacterial strain, extract plasmid (seeing the disclosed data of Qiagen company), in recombinant plasmid transformed e. coli bl21 Rosset cell, coat on the LB solid medium that contains kantlex and paraxin, picking list bacterium colony is put into 37 ℃ of the LB substratum, the 225rpm that contain 100mg/L kantlex and the paraxin activation culture of spending the night.After the activation culture, in 1: 250 ratio bacterium liquid is added in the fresh LB substratum and (to contain 100mg/L kantlex and paraxin), measure OD600 value (Beijing general analyse the logical TU-1800 of instrument company type ultraviolet spectrophotometer), initial OD is transferred unanimity, (37 ℃ of shaking culture, 225rpm) behind the 3h, add 1Mm IPTG, survey the OD value, shaking culture is (42 ℃ again, 225rpm 10h), per hour once surveys the OD value.

Found that the intestinal bacteria that change castor-oil plant homologous gene RcTR1 have higher thermotolerance than not genetically modified intestinal bacteria.The OD value is big more, show that bacterial concentration is big more, be that growing state is good more, as shown in Figure 2, change in time, the growth curve slope of the intestinal bacteria (RcTR1) of commentaries on classics castor-oil plant homologous gene RcTR1 illustrates that obviously greater than non-transgenic intestinal bacteria (contrast) the colibacillary speed of growth and the upgrowth situation that change castor-oil plant homologous gene RcTR1 obviously are better than the non-transgenic intestinal bacteria.

Obtaining and the colibacillary thermotolerance experiment of OsTR1-1, OsTR1-2 of the paddy rice homologous gene OsTR1-1 of embodiment six, AtTR1, OsTR1-2 sequence

1, the clone of paddy rice homologous gene OsTR1-1, OsTR1-2 and obtaining

Extract the total RNA of paddy rice (Invitrogen company's T RIzol reagent) with the TRIzol method, reverse transcription then (promega company reverse transcription test kit) becomes cDNA, respectively according to the design of nucleotide sequence shown in SEQ ID NO:45 (its amino acid sequence coded is seen SEQ ID NO:46), the SEQ ID NO:47 (its amino acid sequence coded is seen SEQ ID NO:48) primer:

OsTR1-1 sequence (SEQ ID NO:45) amplimer:

Upstream primer (SEQ ID NO:49): 5 '-AAACTTTTTTGGGTGATTTGC-3 '

Downstream primer (SEQ ID NO:50): 5 '-CAAATTCTATTGCCCTTGTTCT-3 '

OsTR1-2 sequence (SEQ ID NO:57) amplimer:

Upstream primer (SEQ ID NO:51): 5 '-GGAATTTGGGATGGGCGACCA-3 '

Downstream primer (SEQ ID NO:52): 5 '-AGAGACGCTACTGAAGTAACTAGCTAT-3 '

281-1150 bit sequence (its amino acid sequence coded is seen SEQ ID NO:48) in 71-854 bit sequence (its amino acid sequence coded is seen SEQ ID NO:46), the OsTR1-2 sequence (SEQ ID NO:47) through PCR increases OsTR1-1 sequence (SEQ ID NO:45) from rice cDNA in.Purified pcr product (seeing the disclosed data of Qiagen company) through sequence verification, obtains required sequence.

2, be with the primer of restriction enzyme site according to above-mentioned sequence fragment design of increasing, construction recombination plasmid.

OsTR1-1 sequence (SEQ ID NO:45) band restriction enzyme site amplimer:

Upstream primer (SEQ ID NO:53): 5 '-CGCGGATCCATGGGGGATCATGTTGCGGTGGAT-3 '

Downstream primer (SEQ ID NO:54): 5 '-CCGGAGCTCCTATTGCCCTTGTTCTGGATGAGGT-3 '

OsTR1-2 sequence (SEQ ID NO:47) band restriction enzyme site amplimer:

Upstream primer (SEQ ID NO:55): 5 '-CGCGGATCCATGGGCGACCATGTGGTGGT-3 '

Downstream primer (SEQ ID NO:56): 5 '-CCGGAGCTCGACGCTACTGAAGTAACTAGCTAT-3 '

Through PCR, from the PCR product of step 1, amplify expressed sequence (among the SEQ ID NO:45 among 96-848 bit sequence, the SEQ ID NO:47 291-1147 bit sequence).Purified pcr product (seeing the disclosed data of Qiagen company) is cut with BamH1 and xhol1 enzyme then, and glue is connected with carrier pET28 respectively after reclaiming fragment, obtains recombinant plasmid.Recombinant plasmid is changed in the e. coli jm109, coat on the LB solid medium that contains kantlex,, obtain containing the e. coli jm109 bacterial strain of recombinant plasmid through sequence verification.

3, after recombinant plasmid is bred in the e. coli jm109 bacterial strain, extract plasmid (seeing the disclosed data of Qiagen company), in recombinant plasmid transformed e. coli bl21 Rosset cell, coat on the LB solid medium that contains kantlex and paraxin, picking list bacterium colony is put into 37 ℃ of the LB substratum, the 225rpm that contain 100mg/L kantlex and the paraxin activation culture of spending the night.After the activation culture, in 1: 250 ratio bacterium liquid is added in the fresh LB substratum and (to contain 100mg/L kantlex and paraxin), measure OD600 value (Beijing general analyse the logical TU-1800 of instrument company type ultraviolet spectrophotometer), initial OD is transferred unanimity, (37 ℃ of shaking culture, 225rpm) behind the 3h, add 1mMIPTG, survey the OD value, shaking culture is (42 ℃ again, 225rpm 10h), per hour once surveys the OD value.

Found that two kinds of intestinal bacteria that change paddy rice homologous gene OsTR1-1 and OsTR1-2 have higher thermotolerance than not genetically modified intestinal bacteria.The OD value is big more, show that bacterial concentration is big more, be that growing state is good more, as shown in Figure 3, change in time, the growth curve slope that changes the homogenic intestinal bacteria of paddy rice (OsTR1-1 and OsTR1-2) illustrates that obviously greater than non-transgenic intestinal bacteria (contrast) the colibacillary speed of growth and the upgrowth situation that change paddy rice homologous gene OsTR1-1 and OsTR1-2 obviously are better than the non-transgenic intestinal bacteria.

The colibacillary thermotolerance experiment of obtaining and change beautiful ZmTR1 of the corn homologous gene ZmTR1 of embodiment seven AtTR1

1, the clone of corn homologous gene ZmTR1 and obtaining

Extract the total RNA of corn (Invitrogen company's T RIzol reagent) with the TRIzol method, reverse transcription then (promega company reverse transcription test kit) becomes cDNA, designs primer according to nucleotide sequence shown in the SEQ ID NO:57 (its amino acid sequence coded is seen shown in the SEQ ID NO:58):

Upstream primer (SEQ ID NO:59): 5 '-TTGGGATGGCTGGTGACG-3 '

Downstream primer (SEQ ID NO:60): 5 '-GTGAGCAACTACTGGGGATGTG-3 '

153-1051 bit sequence through PCR increases SEQ ID NO:54 nucleotide sequence from corn cDNA in.Purified pcr product (seeing the disclosed data of Qiagen company) through sequence verification, obtains required sequence.

2, be with the primer of restriction enzyme site according to above-mentioned sequence fragment design of increasing, construction recombination plasmid.

Upstream primer (SEQ ID NO:61): 5 '-CCGGAATTCATGGCTGGTGACGACC-3 '

Downstream primer (SEQ ID NO:62): 5 '-CCCAAGCTTCTATTGCTGTTGCTGCGAC-3 '

Through PCR, from the PCR product of step 1, amplify expressed sequence (158-1018 bit sequence among the SEQ ID NO:54, its amino acid sequence coded are seen SEQ ID NO:58).Purified pcr product (seeing the disclosed data of Qiagen company) is cut with EcoR1 and HindIII enzyme then, and glue is connected with carrier pET28 respectively after reclaiming fragment, obtains recombinant plasmid.Recombinant plasmid is changed in the e. coli jm109, coat on the LB solid medium that contains kantlex,, obtain containing the e. coli jm109 bacterial strain of recombinant plasmid through sequence verification.

3, after recombinant plasmid is bred in the e. coli jm109 bacterial strain, extract plasmid (seeing the disclosed data of Qiagen company), in recombinant plasmid transformed e. coli bl21 Rosset cell, coat on the LB solid medium that contains kantlex and paraxin, picking list bacterium colony is put into 37 ℃ of the LB substratum, the 225rpm that contain 100mg/L kantlex and the paraxin activation culture of spending the night.After the activation culture, in 1: 250 ratio bacterium liquid is added in the fresh LB substratum and (to contain 100mg/L kantlex and paraxin), measure OD600 value (Beijing general analyse the logical TU-1800 of instrument company type ultraviolet spectrophotometer), initial OD is transferred unanimity, (37 ℃ of shaking culture, 225rpm) behind the 3h, add 1mMIPTG, survey the OD value, shaking culture is (42 ℃ again, 225rpm 10h), per hour once surveys the OD value.

Found that the intestinal bacteria that change corn homologous gene ZmTR1 have higher thermotolerance than not genetically modified intestinal bacteria.The OD value is big more, show that bacterial concentration is big more, be that growing state is good more, as shown in Figure 4, change in time, the growth curve slope of the intestinal bacteria (ZmTR1) of commentaries on classics corn homologous gene ZmTR1 illustrates that obviously greater than non-transgenic intestinal bacteria (contrast) the commentaries on classics homogenic colibacillary speed of growth of ZmTR1 corn and upgrowth situation obviously are better than the non-transgenic intestinal bacteria.

Embodiment eight AtTR1 structural researches

Present embodiment adopts PTP to stride the film district, BOR5 strides film district replacement AtTR1 self and strides film district (180-239 amino acids among the SEQ ID NO.2), lack AtTR1 self simultaneously and stride the film district, study AtTR1, replace and to stride the AtTR1 in film district, only contain the thermotolerance that RINGv structure (69-120 among the SEQ ID NO.2, its aminoacid sequence see SEQ ID NO.63) and disappearance are striden the AtTR1 in film district with this.

1, the amplification of protein fragments base sequence

Owing to stride the film district and all derive from Arabidopis thaliana for above-mentioned two kinds, therefore be template with Arabidopis thaliana cDNA, increase respectively above-mentioned two kinds with PCR and to stride the proteic base fragment in film district, simultaneously the base fragment of peptide section that pcr amplification goes out to express peptide section, the 69-120 amino acids of 1-179 amino acids among the SEQ ID NO.2 from Arabidopis thaliana cDNA.

2, the acquisition of vector construction and reorganization bacterium

Cut above-mentioned gene fragment that amplifies and pET28a carrier with EcoRI, Ecl136II enzyme, glue reclaims, and changes escherichia coli DH5a after the connection over to, selects positive colony, extracts plasmid.Plasmid is changed in the intestinal bacteria Rosseta cell, coat on the LB solid medium that contains kantlex and paraxin picking list bacterium colony.So far obtain four kinds of reorganization bacterium altogether:

Contain the reorganization bacterium of AtTR1 full-length proteins, be designated as AtTR1;

AtTR1 self is striden the film district replace with the reorganization bacterium that PTP strides the film district, be designated as AtTR1+PTP;

AtTR1 self is striden the film district replace with the reorganization bacterium that BOR5 strides the film district, be designated as AtTR1+BOR5;

The reorganization bacterium that contains AtTR1 (69-120aa) promptly only contains the RINGv structure, does not have the reorganization bacterium that strides the film district, is designated as AtTR1 (69-120).

3, thermotolerance is measured

Picking list bacterium colony is put into 37 ℃ of the LB substratum, the 225rpm that contain 50mg/L kantlex and the 34mg/L paraxin activation culture of spending the night.After the activation culture, in 1: 100 ratio bacterium liquid is added in the fresh LB substratum and (to contain 50mg/L kantlex and 34mg/L paraxin), 37 ℃, 225rmp were cultivated 2 hours, measure OD600 value (general the analysing in Beijing led to the TU-1800 of instrument company type ultraviolet spectrophotometer) to about 0.2, add IPTG to final concentration 1mM, afterwards with bacterium liquid to 42 ℃, the 225rpm shaking culture, surveyed an OD value in per 1 hour, draw growth curve according to measuring the OD value.Growth curve measurement result such as table 6 and Fig. 5.

Table 6 growth curve measurement result (OD600 value)

By the result as can be seen, the AtTR1 group thermotolerance of expressing full-length proteins is best, apparently higher than AtTR1+PTP, AtTR1+BOR5, AtTR1 (69-120) and empty carrier contrast; Replace AtTR1+PTP, the AtTR1+BOR5 group of striding the film district and AtTR1 (69-120) though group still has certain thermotolerance, be starkly lower than AtTR1 group; The heat-resisting degree of empty carrier contrast is the poorest, can not grow in 42 ℃ hot environment substantially.

Embodiment nine AtTR1 paddy rice homologous protein OsTR1-1 structural researches

Present embodiment adopts PTP to stride the film district, BOR5 strides film district replacement OsTR1-1 self and strides film district (SEQ ID NO.45 sequence synthetic proteins 145-203 amino acids), lack OsTR1-1 self simultaneously and stride the film district, study OsTR1-1, replace and to stride the OsTR1-1 in film district, only contain the thermotolerance that RINGv structure and disappearance are striden the OsTR1-1 in film district (SEQ ID NO.45 sequence synthetic proteins 32-81aa, its aminoacid sequence is seen SEQ ID NO.66) with this.

Experimental program finally obtains four kinds of reorganization bacterium referring to embodiment eight:

Contain the reorganization bacterium of OsTR1-1 full-length proteins, be designated as OsTR1-1;

Contain and OsTR1-1 self is striden the film district replace with the reorganization bacterium that PTP strides the film district, be designated as OsTR1-1+PTP;

Contain and OsTR1-1 self is striden the film district replace with the reorganization bacterium that BOR5 strides the film district, be designated as OsTR1-1+BOR5;

The reorganization bacterium that contains OsTR1-1 (32-81aa) promptly only contains the RINGv structure, does not have the reorganization bacterium that strides the film district, is designated as OsTR1-1 (32-81).

IPTG 1mM, 42 ℃, 225rpm shaking culture 10 hours, growth curve measurement result such as table 7 and Fig. 6.

Table 7 growth curve measurement result (OD600 value)

By the result as can be seen, the OsTR1-1 group thermotolerance of expressing full-length proteins is best, apparently higher than OsTR1-1+PTP, OsTR1-1+BOR5, OsTR1-1 (32-81) and empty carrier contrast; Replace OsTR1-1+PTP, the OsTR1-1+BOR5 group of striding the film district and OsTR1-1 (32-81) though group still has certain thermotolerance, be starkly lower than OsTR1-1 group; The heat-resisting degree of empty carrier contrast is the poorest, can not grow in 42 ℃ hot environment substantially.

Embodiment ten AtTR1 corn homologous protein ZmTR1 structural researches

Present embodiment adopts PTP to stride the film district, BOR5 strides film district replacement ZmTR1 self and strides film district (SEQ ID NO.57 sequence synthetic proteins 175-234 amino acids), lack ZmTR1 self simultaneously and stride the film district, study ZmTR1, replace and to stride the ZmTR1 in film district, only contain the thermotolerance that RINGv structure (SEQ ID NO.57 sequence synthetic proteins 68-118 amino acids) and disappearance are striden the ZmTR1 (its aminoacid sequence is seen SEQ ID NO.65) in film district with this.

Experimental program finally obtains four kinds of reorganization bacterium referring to embodiment eight:

Contain the reorganization bacterium of ZmTR1 full-length proteins, be designated as ZmTR1;

Contain and ZmTR1 self is striden the film district replace with the reorganization bacterium that PTP strides the film district, be designated as ZmTR1+PTP;

Contain and ZmTR1 self is striden the film district replace with the reorganization bacterium that BOR5 strides the film district, be designated as ZmTR1+BOR5;

The reorganization bacterium that contains ZmTR1 (68-118aa) promptly only contains the RINGv structure, does not have the reorganization bacterium that strides the film district, is designated as ZmTR1 (68-118).

IPTG 1mM, 42 ℃, 225rpm shaking culture 10 hours, the growth curve measurement result sees Table 8 and Fig. 7.

Table 8 growth curve measurement result (OD600 value)

By the result as can be seen, the ZmTR1 group thermotolerance of expressing full-length proteins is best, apparently higher than ZmTR1+PTP, ZmTR1+BOR5, ZmTR1 (68-118) and empty carrier contrast; Replace ZmTR1+PTP, the ZmTR1+BOR5 group of striding the film district and ZmTR1 (68-118) though group still has certain thermotolerance, be lower than ZmTR1 group; The heat-resisting degree of empty carrier contrast is the poorest, can not grow in 42 ℃ hot environment substantially.

Embodiment 11 crosses the thermotolerance of the Arabidopis thaliana of expressing AtTR1, OsTR1-2, ZmTR1, RcTR1 gene and critical section thereof and identifies

With AtTR1 gene (SEQ ID NO:1), OsTR1-2 gene (SEQ ID NO:47), ZmTR1 gene (SEQ IDNO:57), RcTR1 gene (SEQ ID NO:39) and in the crucial separately peptide section of coding SEQ ID NO:63, SEQ ID NO:67, SEQ ID NO:65, the coding nucleotide sequence of SEQ ID NO:64 picked out change in the Arabidopis thaliana, the thermotolerance of AtTR1, OsTR1-2, ZmTR1, RcTR1 gene and crucial peptide fragment gene Arabidopis thaliana thereof is changeed in research.

Carrier construction method utilizes inflorescence dip method arabidopsis thaliana transformation referring to embodiment two.Detailed step is as follows:

1, picking contains SEQ ID NO:1, SEQ ID NO:47, SEQ ID NO:57, SEQ ID NO:39, the Agrobacterium that reaches the overexpression recombinant plasmid of SEQ IDNO:63, SEQ ID NO:67, SEQ ID NO:65, SEQ ID NO:64 is inoculated in and contains 20mg/L Str, 50mg/L Kan, in the LB liquid nutrient medium of 40mg/L Rif, 28 ℃ are shaken the bacterium back of spending the night and collect thalline, be resuspended in the MS liquid nutrient medium that contains 0.01% tensio-active agent silwet-77 to OD600=0.4-0.6,28 ℃ are shaken bacterium 1-2h, and bacterium liquid is stand-by.

2, the inflorescence that grown of the Arabidopis thaliana that will cultivate 60 days is cut, soaked inflorescence 2 minutes with the Agrobacterium bacterium liquid that contains recombinant plasmid, secretly cultivated afterwards 48 hours, the Arabidopis thaliana seedling after dark the cultivation can move into the normal illumination ambient growth, and the pod that grows subsequently is transgenosis T0 for seed.

3, with the seed plantation of results, grow to about 50 days, identify, obtain following positive plant of expressing by PCR:

The transgenic arabidopsis that contains AtTR1 (SEQ ID NO:1) is designated as AtTR1,

The transgenic arabidopsis that contains crucial peptide section (the SEQ ID NO:63) encoding sequence of AtTR1 gene is designated as R-AtTR1,

The transgenic arabidopsis that contains OsTR1-2 (SEQ ID NO:47) is designated as At-OsTR1-2,

The transgenic arabidopsis that contains crucial peptide section (the SEQ ID NO:67) encoding sequence of OsTR1-2 gene is designated as At-R-OsTR1-2,

The transgenic arabidopsis that contains ZmTR1 (SEQ ID NO:57) is designated as At-ZmTR1,

The transgenic arabidopsis that contains crucial peptide section (the SEQ ID NO:65) encoding sequence of ZmTR1 gene is designated as At-R-ZmTR1,

The transgenic arabidopsis that contains RcTR1 (SEQ ID NO:39) is designated as At-RcTR1,

The transgenic arabidopsis that contains crucial peptide section (the SEQ ID NO:64) encoding sequence of RcTR1 gene is designated as At-R-RcTR1,

After the transfer-gen plant maturation, it is standby to collect seed.

4, the seed heat stress is sprouted experiment

The MS substratum for preparing (prescription sees Table 8, and pH transfers to 5.8 with KOH) divides behind the high pressure steam sterilization to install in the culture dish.Change on the substratum with 2mL sterilized water suspension seed after the culture medium solidifying, treat that seed evenly after planting removes unnecessary sterilized water, open the ware lid, in gnotobasis, place 1h, seal to surface drying.

Experiment is handled and is divided two groups, first group is the heat shock group: culture dish after planting is put into (50 ℃ of culturing room, intensity of illumination 6000～8000lx, 16h/8h light dark period, relative humidity 70%) heat shock handled 2 hours, move to (22 ℃ of normal temperature environment growths afterwards, intensity of illumination 6000～8000lx, 16h/8h light dark period, relative humidity 70%), observe every day and sprout and other phenotypes, the statistics germination rate.Second group is heat shock control group not: culture dish after planting is put into culturing room's (22 ℃, intensity of illumination 6000～8000lx, 16h/8h light dark period, relative humidity 70%) growth, observes every day and sprout and other phenotypes, the statistics germination rate.Experimental result sees Table 9.

Table 8 MS culture medium prescription

Germination rate after the heat shock of table 9 Arabidopis thaliana seed

	After 50 ℃ of heat shocks 22 ℃ the 5th day	The heat shock growth regulation is 5 days
			AtTR1	74％	100％
R-AtTR1	62％	100％
			At-OsTR1-2	56％	96％
At-R-OsTR1-2	40％	98％
			At-ZmTR1	60％	100％
At-R-ZmTR1	42％	98％
			At-RcTR1	48％	100％
At-R-RcTR1	30％	100％
			WT
	6％	98％

No matter found that after handling by heat shock, the sprouting of Arabidopis thaliana seed has been subjected to influence, be transgenosis type or non-transgenic contrast, and the germination rate after the heat shock all is lower than the normal temperature growth group that not heat shock is handled.Find by contrast, change the influence degree minimum that AtTR1, OsTR1-2, ZmTR1, transgenic arabidopsis AtTR1, the At-OsTR1-2 of RcTR1 complete sequence, At-ZmTR1, At-RcTR1 are subjected to heat shock over to, germination rate 74%-48%; Transgenic arabidopsis R-AtTR1, the At-R-OsTR1-2, At-R-ZmTR1, the At-R-RcTR1 that change AtTR1, OsTR1-2, ZmTR1, RcTR1 pass key sequence over to are subjected to the influence degree of heat shock less, contrast than non-transgenic, still has certain thermotolerance, germination rate 62%-30%, and the germination rate of non-transgenic type WT only is 6%.

Arabidopis thaliana after the heat shock processing is continued as for 22 ℃ intensity of illumination 6000～8000lx, 16h/8h light dark period, grow in relative humidity 70% environment, found that, along with time lengthening, the non-transgenic contrast can't grow green basic leaf, and the seedling look is yellow or white, and is final dead; And the transgenosis type all can grow green basic leaf, and upgrowth situation is good, can blossom and bear fruit.

Explanation, no matter AtTR1 gene, OsTR1-2 gene, ZmTR1 gene, RcTR1 gene be that complete sequence (SEQ ID NO:1, SEQ ID NO:47, SEQ ID NO:57, SEQ ID NO:39) still closes key sequence (SEQ ID NO:63, SEQ ID NO:67, SEQ ID NO:65, SEQ ID NO:64) and all can improve plant to the pyritous tolerance, makes its sprouting, growth keep good order and condition.

Embodiment 12 AtTR1, the proteic E3 ubiquitin ligase of OsTR1-1, ZmTR1 activity

Preparation 20ul reaction system: 1ug E1 albumen, 2ug E2 albumen, 4ug AtTR1 albumen (or OsTR1-1 albumen or ZmTR1 albumen), 0.1mmol/L ubiquitin (Ub), 2mmol/L ATP, 5mmol/L MgCl2,50mmol/L Tris, pH7.5.Under 30 ℃ of conditions, react.Reaction adds 6 * SDS-PAGE sample-loading buffer stopped reaction after the 90min, carries out the SDS-PAGE electrophoresis after boiling 5min.

Electrotransfer uses the monoclonal antibody (available from Santa Cruz) of anti-Ub and alkaline phosphatase (AP) link coupled mouse-anti mouse two anti-(available from Promega) to carry out Western Blot detection to poly(vinylidene fluoride) (PVDF) film subsequently behind the electrophoresis.

Result such as Fig. 8,9,10 show, in the external albumen test, AtTR1, OsTR1-1, ZmTR1 albumen are under suitable E1, E2, ATP and Ub acting in conjunction, Ub can be connected with AtTR1, OsTR1-1, ZmTR1 albumen, and formation poly chain, as can be seen from Figure, add simultaneously and occur many barss band in the swimming lane of E1, E2, ATP and Ub, and contrast is without any the signal band, show that AtTR1, OsTR1-1, ZmTR1 albumen have ubiquitin E3 ligase enzyme activity, the formation of energy catalysis monomer or poly ubiquitin.

By above-mentioned example as seen, a-protein tTR1 of the present invention and the homologous protein in plants such as corn, paddy rice, castor-oil plant thereof all have the plant of raising or the stable on heating function of microorganism.Discover, this proteinoid all contains the zinc fingers of RINGv type, and all belong to the E3 ligase enzyme, by research of the present invention, found out the critical area that this proteinoid plays a role---RINGv zinc fingers, the E3 ligase enzyme albumen that contains this structure can make the thermotolerance of plant, microorganism increase, and the albumen of removing this structure then loses fully and improves plant, the stable on heating effect of microorganism.In addition also this type of proteic film district of striding is studied, discover the thermotolerance that membrane structure can better improve plant, microorganism of striding of albumen self, but after replacing to other and striding membrane structure, still have certain thermotolerance, and heat-resisting degree is higher than the non-transgenic type.As fully visible, the zinc fingers of RINGv is only the key structure of this proteinoid performance effect of AtTR1, promptly has N '-CRICQE X _7-45PCAC X ₆AHR X ₁CVQ X _13-27-C ' or further have N '-CRICQEED X _3-20NL X _3-20PCAC X ₂SLK X ₁AHR X ₁CVQRWC X _10-24The peptide section of-C ' (wherein X is an arbitrary amino acid, and subscript is represented the quantity of amino-acid residue) is to bring stable on heating crucial peptide section.The gene of this class coding thermostable protein provided by the invention and coded protein thereof can bring the polypeptide of heat-resisting function and these genes, protein and the polypeptide purposes in the thermotolerance that improves plant and microorganism in addition.For this area provides new alternative gene, has good application prospects.

Claims (43)

1. coding improves plant or the stable on heating proteinic gene of microorganism, it is characterized in that containing the nucleotide sequence of following peptide section of encoding:

N '-CRICQE X _7-45PCAC X ₆AHR X ₁CVQ X _13-27-C ', wherein X is an arbitrary amino acid, the quantity of following target numeral amino-acid residue.

2. improve plant or the stable on heating proteinic gene of microorganism according to the described coding of claim 1, it is characterized in that described peptide section is: N '-CRICQEED X _3-20NL X _3-20PCAC X ₂SLK X ₁AHR X ₁CVQRWC X _10-24-C ', wherein X is an arbitrary amino acid, the quantity of following target numeral amino-acid residue.

3. improve plant or the stable on heating proteinic gene of microorganism according to the described coding of claim 1, it is characterized in that described protein is ubiquitin ligase.

4. improve plant or the stable on heating proteinic gene of microorganism according to the described coding of claim 1, it is characterized in that: described albumen is transmembrane protein.

5. improve plant or the stable on heating proteinic gene of microorganism according to the described coding of claim 1, it is characterized in that: described albumen also contains strides the film district.

6. improve plant or the stable on heating proteinic gene of microorganism according to the described coding of claim 4, it is characterized in that: described albumen also contains 1～6 and strides the film district.

7. improve plant or the stable on heating proteinic gene of microorganism according to the described coding of claim 4, it is characterized in that: described albumen also contains 2～3 and strides the film district.

8. improve plant or the stable on heating proteinic gene of microorganism according to the described coding of claim 7, it is characterized in that: described structure of striding the film district is N '-A X _2-6CRS X _2-8LIL X _2-4LL X _1-4LR X _1-10-C ' or N '-L X _2-4R X _1-5GFLL X _1-7YIMAW X _1-15At least a shown in the-C ', wherein X is an arbitrary amino acid, subscript is represented the quantity of amino-acid residue.

9. improve plant or the stable on heating proteinic gene of microorganism according to the described coding of claim 4, it is characterized in that: described albumen also contains signal peptide.

10. claim 9 is stated coding raising plant or the stable on heating proteinic gene of microorganism, and it is characterized in that: described signal peptide is the film localization signal peptide.

11. improve plant or the stable on heating proteinic gene of microorganism according to each described coding of claim 1～4, it is characterized in that: described gene source is in the genome of plant.

12. coding according to claim 5 improves plant or the stable on heating proteinic gene of microorganism, it is characterized in that: described plant is Arabidopis thaliana, paddy rice, corn or castor-oil plant.

13. improve plant or the stable on heating proteinic gene of microorganism according to each described coding of claim 1～12, it is characterized in that: (1): described gene has the nucleotide sequence shown in SEQ ID NO:39, SEQ ID NO:1, SEQ ID NO:45, SEQ ID NO:47 or the SEQ ID NO:57;

Perhaps (2): described gene has in (1) described arbitrary nucleotide sequence through replacing, lack or add the derive nucleotide sequence of gained of at least one Nucleotide, and the protein of coding with same or analogous function.

14. coding according to claim 11 improves plant or the stable on heating proteinic gene of microorganism, it is characterized in that: (1): described gene has, the nucleotide sequence shown among among among among the SEQ ID NO:57 the 158th～1018, SEQ ID NO:45 the 96th～848, SEQ ID NO:47 291～1127 or the SEQ ID NO:39 the 68th～946;

Perhaps (2): described gene has in (1) described arbitrary nucleotide sequence through replacing, lack or add the derive nucleotide sequence of gained of at least one Nucleotide, and the protein of coding with same or analogous function.

15. improve plant or the stable on heating protein of microorganism, it is characterized in that containing following peptide section:

N '-CRICQE X _7-45PCAC X ₆AHR X ₁CVQ X _13-27-C ', wherein X is an arbitrary amino acid, subscript is represented the quantity of amino-acid residue.

16. raising plant according to claim 15 or the stable on heating protein of microorganism, it is characterized in that: the structure of described peptide section is: N '-CRICQEED X _3-20NL X _3-20PCAC X ₂SLK X ₁AHR X ₁CVQRWC X _10-24-C ', wherein X is an arbitrary amino acid.

17. raising plant according to claim 15 or the stable on heating protein of microorganism is characterized in that: described albumen also contains strides the film district.

18. raising plant according to claim 17 or the stable on heating protein of microorganism is characterized in that: described albumen also contains 1～6 and strides the film district.

19. raising plant according to claim 18 or the stable on heating protein of microorganism is characterized in that: described albumen contains 2～3 and strides the film district.

20. raising plant according to claim 17 or the stable on heating protein of microorganism is characterized in that: described structure of striding the film district is N '-A X _2-6CRS X _2-8LIL X _2-4LL X _1-4LR X _1-10-C ' or N '-L X _2-4R X _1-5GFLL X _1-7YIMAW X _1-15At least a shown in the-C ', wherein X is an arbitrary amino acid, the quantity of following target numeral amino-acid residue.

21. raising plant according to claim 15 or the stable on heating protein of microorganism is characterized in that: described protein is ubiquitin ligase.

22. raising plant according to claim 15 or the stable on heating protein of microorganism, it is characterized in that: described albumen also contains signal peptide.

23. claim 22 described raising plant or the stable on heating protein of microorganism is characterized in that: described signal peptide is the film localization signal peptide.

24. according to each described raising plant of claim 15～23 or the stable on heating protein of microorganism, it is characterized in that: described protein source is in plant.

25. according to each described raising plant of claim 15～24 or the stable on heating protein of microorganism, it is characterized in that: (1) described protein has by the coded aminoacid sequence that obtains of nucleotide sequence shown in the 68th～946 among 291～1127 among the 96th～848 among the 158th～1018 among SEQ ID NO:1, the SEQ ID NO:57, SEQ ID NO:45, SEQ ID NO:47 or the SEQ ID NO:39;

Perhaps (2): described gene has process replacement in (1) described arbitrary aminoacid sequence, lacks or add the aminoacid sequence of at least one amino acid derived gained, and has same or analogous function.

26. coding each described raising plant of claim 15～25 or the stable on heating proteinic gene of microorganism.

27. the peptide section is characterized in that: contain N '-CRICQE X _7-45PCAC X ₆AHR X ₁CVQ X _13-27Structural domain shown in the-C ', wherein X is an arbitrary amino acid, subscript is represented the quantity of amino-acid residue.

28. peptide section according to claim 27 is characterized in that: contain N '-CRICQEED X _3-20NL X _3-20PCAC X ₂SLK X ₁AHR X ₁CVQRWC X _10-24Structural domain shown in the-C ', wherein X is the quantity that is designated as amino-acid residue under the arbitrary amino acid.

29. peptide section according to claim 27 is characterized in that: derive from plant.

30. peptide section according to claim 27 is characterized in that: described plant is Arabidopis thaliana, paddy rice, corn or castor-oil plant.

31. according to claim 27 or 28 described peptide sections, it is characterized in that: described peptide section can form zinc fingers.

32. according to claim 27 or 28 described peptide sections, it is characterized in that: described peptide section can be given protein and be improved plant or the stable on heating function of microorganism.

33., it is characterized in that: have following peptide sequence according to each described peptide section of claim 27～32:

(1): the aminoacid sequence shown in SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66 or SEQ ID NO:67;

Perhaps, process replaces, lacks or add the aminoacid sequence of at least one amino acid derived gained in (1) described arbitrary amino acid sequence of polypeptide, and has same or analogous function.

34. the nucleotide sequence of each described peptide section of coding claim 27～33.

Nonrefractory proteinic genetic modification will be the method for the gene of coding thermostable protein 35. will encode, it is characterized in that having following steps: the nucleotide sequence of each described peptide section of the claim 27～33 of will encoding is added in the nonrefractory proteinic gene of coding or replaces the nonrefractory proteinic gene of coding one section nucleotide sequence wherein, described interpolation or replacement are to occur between the codon, so that whole improved gene still has the ability of coding whole protein.

36. method according to claim 35, it is characterized in that the one section nucleotides sequence that is replaced in the nonrefractory proteinic gene of described coding classify as the coding Zinc finger domain nucleotide sequence.

37. according to claim 35 or 36 described methods, it is characterized in that: described nonrefractory protein is ubiquitin ligase.

38. improve plant heat resistance property or prepare the method for high heat resistance plant, it is characterized in that: may further comprise the steps:

A, each described gene of claim 1～14 operationally is connected in expression regulation sequence on the carrier after, formation can be expressed the recombinant vectors of this gene;

B, change the recombinant vectors in the step (1) over to vegetable cell;

C, obtain transformant through screening, then transformant is cultivated into transfer-gen plant or its offspring, described offspring comprises plant seed or plant tissue.

39. contain the carrier that each described coding of claim 1～14 improves plant or the stable on heating proteinic gene of microorganism.

40. according to the described carrier of claim 39, it is characterized in that: described carrier is an expression vector.

41. according to the described carrier of claim 40, it is characterized in that: described expression vector is a carrier for expression of eukaryon.

42. contain the host cell that each described coding of claim 1～14 improves plant or the stable on heating proteinic gene of microorganism.

43. according to the described host cell of claim 40, it is characterized in that: described host cell is vegetable cell or microorganism.

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